In the early 1970's, a scientist at the
University of Oregon by the name of Dr. George
Streisinger determined that the zebrafish is a wonderful model for studying vertebrate
development and genetics. Since he began using them in his research, zebrafish embryos
have become very popular worldwide as a means of understanding how not only fish, but all
vertebrates including people, develop from the moment that sperm fertilizes an egg. The
eggs are clear and develop outside of the mother's body, allowing scientists to watch a
zebrafish egg grow into a newly formed fish under a microscope. The scientists watch while
the cells divide and form different parts of the baby fish's body. In the development span
of 2-4 days, some form to make the eyes; others, the heart, the liver, the stomach, the
skin, the fins, etc. until the fish is complete and ready to begin it's new life.
Scientists will occasionally move a cell to another spot to see if it will still go to
form the same part of the body as it is known to do in other embryos or if it will do
something different. Occasionally a cell is removed or destroyed to see what the result is
to the fish once it has developed. This is how scientists are discovering the causes of
birth defects in human children and it's how they are trying to find a way to prevent
these birth defects by understanding why they happen and what original cells are involved.
So, this little obscure fish of the Ganges is helping us to learn about how all
vertebrates develop and why sometimes things go wrong in that development to cause birth
defects and other health problems. It is serving a very important role in our
understanding and some day it may play a huge role in overcoming these things.

Zebrafish are vertebrates. Like humans, they have a backbone. This means that they are more closely
related to humans than commonly used invertebrate models such as insects and worms (Drosophilia
- fruit flies and Caenorhabditis elegans - nematodes) which do not have backbones.
Because zebrafish are more closely related to humans, they are more likely to be similar
to them in many biological traits than a more distantly related organism. These biological
traits would include genes, developmental processes, anatomy, physiology, and behaviors.
This is an advantage that invertebrate lab animals do not share with humans. The
invertebrates are more appropriately used in comparisons at the cellular or biochemical
level of organization where they share many features with humans.

No single model is perfect, but zebrafish
have features that make them easy to maintain, manipulate, and
observe in the lab. They do well in many environments, and their small size, their ability
to be kept together in large numbers, and the ease with which they can be bred makes them
a favored model. Breeding and getting eggs from the zebrafish is relatively easy. Their
eggs are externally fertilized, produced regularly in large numbers, and are non-adhesive.
Their embryos develop rapidly, and are clear throughout their development. Their embryos
are also smaller than many vertebrate embryos and contain smaller numbers of cells. It is
easier to trace the development of individual cells.

Females lay large quantities of eggs. For many types of genetic analysis you need to look
at many different embryos at many different stages to understand what the problem is with
a given mutation.

The embryos develop outside the mother's body, so you can have easy access to them. In contrast,
mouse embryos develop inside the mother, and you have to kill the mother to get at them.
This would have to be done at each of the stages of development you want to look at. Once
you do this, of course, the embryos die as well as the mother, so you are very limited in
the types of experiments you can do.

Zebrafish embryos are transparent. This means you can watch development as it happens in living embryos.
You can see internal organs, such as the brain, heart, blood, muscles, etc. In addition,
you can monitor the behavior of single cells in live embryos and watch the cells divide
and through dyes, trace where each cells "daughters" go in making up the
complete organism. It is not possible to achieve this resolution with other systems.

The embryos develop quickly. They go from a single cell to something that is
recognizable as a tiny fish within 24 hours. Mice take 21 days.

You can physically manipulate the embryos. By this I mean you can transplant single cells or groups of
cells into host embryos. This kind of experiment is performed frequently to analyze the
behavior of cells at different stages, or to ask how mutant cells behave in wildtype
embryos. This can give us a lot of information about how certain gene products act.
In addition, fertilization of the egg can be manipulated so
that the embryo contains only it's mother's genes. This is done by exposing the sperm to
ultraviolet light which destroys the genes it contains from the male. This allows
scientists to study recessive mutations since the characteristics and defects are
inherited from only one parent.

There is a large community of researchers willing to share their knowledge of the more specific areas of zebrafish
research.

We can't do research on humans so we find models that mimic the human, do the research
using zebrafish, and then try to figure out how
to extrapolate the data to humans... not an easy task. We are really never sure how
exactly the data will match what we would have found if we use humans. Now that we know
the genome of the human and are working on many other organisms, that process is getting
easier. For example, if we find some gene altered in the zebrafish due to exposure to a
toxicant or from a disease, then we can use the database to search for a similar gene in
humans. We also now know that there is considerable conservation of pathways across
species. Zebrafish are vertebrates. This means that they are more closely related to
humans than invertebrates. By virtue of their being more closely related to humans, they
are more likely to be similar in any biological trait than is a more distantly related
organism.

Some zebrafish mutants are known to develop
and duplicate certain conditions and diseases common to humans. In studying their more
simplified make up, it is frequently possible to determine what genes are involved and
then compare them to the equivalent genes within the human genome.

Although zebrafish and humans are obviously
very different, their embryonic development is remarkably similar. Furthermore, it is
becoming more and more clear that all vertebrates follow an evolutionarily-conserved
developmental program. This conservation extends even to the molecular level--where
similar genes perform similar functions in many different species. You cannot do the kind
of experiments in humans that are outlined above, and which are necessary to understand a
given biological process, and how genes act to make that process work.

Examples:

There are many zebrafish mutants with defective blood (studied primarily by Len Zon's
lab - Massachusetts General Hospital) or defects in heart development (Mark Fishman's Lab
- Massachusetts General Hospital, Didier Stainier's Lab - University of California, SF, or
Debbie Yelon's lab - Skirball Institute, NYU School of Medicine). It is likely that blood
function and heart development in zebrafish are similar to blood function and heart
development in people. Some of these mutations might mimic human syndromes, and
understanding them will provide us with valuable insight as to the underlying problem.
Such an understanding could lead to new treatments. In addition, many of the proteins
involved in early development are critical in later life. Defects in these proteins, or in
the regulation of their expression, can lead to tumors later in life.

Features of the zebrafish strongly favor studying embryology
and collecting and analyzing mutations and comparing them to other
vertebrates. There are invertebrates that are equally or better suited for these types of
studies, but they have very different anatomy patterns of development. These and other
approaches allow a problem to be studied from many different experimental points of view,
resulting in a better understanding of it.

There is now a large base of established knowledge on the developmental biology and genetics of the
zebrafish. Many useful genetic techniques and
staining reagents have been developed that allow mutations to be more easily found,
studied, and mapped.

You can do genetic analysis. This means you can generate mutations, and identify
genes required for a wide variety of biological processes. Genes direct synthesis of
proteins, which do all the work in a cell. A mutation is a change of DNA that disrupts the
resulting protein. Often, a defective protein will disrupt an essential biological
process--and we can learn a lot about that process by analyzing how it can be screwed up.
We can also learn much about the role of the normal protein by understanding what goes
wrong when that protein is defective.

There is a detailed genetic map of the
zebrafish genome.
This facilitates the identification of proteins disrupted by mutations. The genome is
currently being sequenced and is due to be completed by the end of next year. This will
make the process much easier. It will also facilitate evolutionary comparisons of the
zebrafish genome with the mouse and human genomes.

You can make transgenic fish--that means introducing foreign DNA in a heritable
manner. This is crucial for analysis of gene regulation, and also for gene function.

Expense. Fish are cheaper to maintain than mice, but more expensive than flies--another
powerful model organism.

Disadvantages:

They require water systems to maintain them

They are not mammals and are not as closely related to humans as a mouse is.

Reverse genetics has not been worked out for zebrafish as it has in the mouse.

No way of targeting mutations. In mice, for example, you can "knock out" a
gene if you have the sequence, and ask why it is needed. In zebrafish, we create
random mutations and look for specific defects. Then we have to go and find what sequence
is responsible for the defect.

Fly (Drosophila) genetics is much more powerful than fish genetics, because many
genetic tools and tricks have been designed over the course of the last 100 years. We need
to catch up.

Males are slender and torpedo-shaped,
usually with gold on their belly, ventral fin, pelvic fins, and pectoral fins, although
this isn't always true... especially in the Long Fin variety. Males can also be identified
by the fact that they tend to chase the females early in the morning before they breed.
Females are fat when filled with eggs and do not usually have gold on their undersides (or
very little of it, if any) .

These are barbels, a
whiskery-like sensory organ, and are found on both males and females. The
function of the barbels is 1) chemosensory and 2) for disturbing bottom debris
in order to locate food. Barbels are common to fish
living at the bottom of eutrophic waterways where visual location of food is not
easy. The catfish are perhaps the best known example of a fish group having
barbels and they live at the bottom of deepish, frequently dirty, rivers and
water impoundment's.

The zebrafish is a small, hardy fish,
about 1" to 1½" long that many people have in their small aquariums
at home. The standard "wildtype" variety is somewhat clear-colored with black
stripes that run lengthwise down its body. For many years they have been bred by fish
hobbyists for home aquariums.

There are several different
varieties of zebrafish... long finned, short finned (the natural wild type), striped
(natural) and speckled, as well as albino and "pink" and "blue." All
are merely genetic variants of the same species. They can all interbreed and you can do
genetic experiments to determine dominance of the various traits. (i.e., are long fins
dominant over short fins? etc.)

The easiest way to obtain your
stock of fish would be to try the local pet stores. They should have normal looking
wildtype fish and they may have some variants that you could use although it is unlikely
they would have a true albino (no dark pigment and pink eyes). Your most likely choices
would be: a) leopard, a recessive with spots instead of stripes; b) long fin, a dominant
with long flowing fins (a common type of mutant in cultured tropical fish); and c) brass,
a dark pigment-lacking fish except for its eyes which are dark. It is a recessive. You can
often get long fin in combination with one of the other two mutants. There are also a
bunch of other mutants that are infrequently seen in the pet stores. There are hundreds of
them in labs, but most of them are embryonic lethals and several pigmentation mutants. I
would not recommend using lab strains if you can avoid it because they are very inbred and
less robust and viable.

First of all, zebras lay eggs every
day! They eat most of their own eggs as a way of recycling the protein that is lost from
producing the eggs. Feed your zebrafish LOTS of high protein foods. The best is LIVE brine
shrimp but frozen will also work well. Cichlid food is higher in protein than other foods
for fish, too. READ the nutrition information and pick foods high in protein. Do not feed
more than they can consume, though, since excess food contaminates the water. Feed them
well for at least one week or until the females get fat with extra eggs.

Very tiny baby zebrafish in large
facilities eat even tinier living organisms called paramecia that live in water. These
paramecia are grown in jars and are fed to the fish by squirting hundreds of them from an
eye dropper into their water. You can't see them very well. It just looks like cloudy
water in the eye dropper. In general, live food is best for baby fish. It is easier for
them to digest. Your fish will not have to eat until they are floating up off the bottom,
though. Hobbyists traditionally use infusoria, a mix of ciliated microscopic organisms
that can be grown in cultures of boiled grass or hay. For details see a hobbyist book for
those instructions. You can buy an infusoria culture from the pet store in either tablet
or powder form. The directions for cultivating and feeding the fry will be on the package.
Be sure to explain to the person at the pet store that you want to feed live
micro-organisms to baby egg-layers. Another possible food for both the fish and a ciliate
culture is the yolk of a boiled egg after it has been pushed through a small size mesh
(usually an old nylon stocking). People who have zebrafish at home frequently buy and feed
other packaged foods bought from the pet store instead of raising their own. Try to stay
away from artificial substitutes since they tend to pollute the water too easily. As the
fish get bigger and become adults, they eat live baby brine shrimp which are a little
larger than paramecia, but are still hard to see because of their small size. You should
be able to buy frozen baby brine shrimp or eggs to hatch from a pet store. Be sure not to
over feed this and siphon out any left over after 30 minutes. Otherwise it will decay and
the bacteria will make your baby fish sick.

* We are having trouble raising
zebrafish. We have had quite a bit of success getting the eggs to hatch, but we are
unable to keep them alive for more than 24 hours afterwards. We think it's because there isn't any food
for them to eat. We've tried feeding them crushed flake food and have even experimented
with vinegar eels, but still no success. One student who had a huge amount of success
raising the fish used just crushed flake food. What do you think is happening?

If your fish are
hatching but die soon afterward there are a number of possibilities. Some of them
might be best explored with the use of a microscope with a high power magnifying lens. The
baby fish do not really eat much until they develop their gas bladders and can float up
into the water and swim around easily. It is at this time they chase down small prey items
(like paramecia) and eat them. Even if they do not eat, it will take several days for them
to die of starvation. It sounds more likely to me that something else is going on. We have
found that vinegar eels (which we also feed our older fish) do not work well as a first
food. We use paramecia. It may be that the baby fish may not able to deal with eating the
vinegar eels, but some people here at the UO think that the problem is that there is too
much transfer of the vinegar (a weak acid) causing their water to become too acid. The
details of this issue are unresolved, but they have not worked well for us as a first
food.

Another problem we have had is the presence of a small ciliate
(coleps) that will kill and eat the baby zebrafish. You can usually tell if you have this
problem if the number of baby fish decreases very rapidly. The bodies would be eaten up
rapidly and completely. It would appear as if someone had removed the baby fish.

There is also the possibility that the water
you are raising the fish in may be contaminated with some chemical that is causing their
death. Alternatively there may be either fungus or bacteria growing on dead fish or
uneaten food that could cause the fish to die. The water should always be as clear as possible and
everything dead should be removed. If you suspect a chemical problem with the water, try a
different source of water.

The success with the crushed flake food can result in two ways:

1) the flakes may be getting crushed to a
small enough size for the fish to eat directly, or

2) the flake food may be decomposing, thereby
feeding bacteria. The bacteria in turn may be eaten by a variety of ciliates or
protozoans, which in turn are food for the baby fish.

By observing your baby fish, the water they
live in, and what, if anything, they are
eating, you should be able to tell which of these possible scenarios are occurring in your
particular case.

The easiest thing you could try would be an
infusoria culture. Details can be found in many aquarium books, but it is basically made
by boiling some clean grass in water letting it cool down and set with the top open for
several days to weeks until a culture of bacteria and protozoans develops. You could
probably speed this process up by adding some old aquarium or pond water.

No. Frozen brine shrimp are considered
a non-optimal food for a number of reasons. The freezing process damages the shell of the
shrimp resulting in the loss of nutrients. The shrimp will become food for bacteria
or fungus, both bad. The frozen brine shrimp are dead so they will start rotting
immediately and sink to the bottom where they will be a less attractive food item for the
fish. Instead, you should use live baby brine shrimp that you hatch yourself from eggs
(cysts actually). These will swim around a bit, eliciting interest from the fish, and will
provide a fresh source of food.

If a brine shrimp can't fit down the
baby fish's throat they won't be able to eat it. What we usually do is to continue to feed
the baby fish paramecia in addition to the live brine shrimp at least for a while. One way
you can tell if the baby fish are eating the brine shrimp is that their bellies will turn
red from the brine shrimp in their stomachs. Ideally, you should not feed more brine
shrimp than the fish can eat in a short period of time... maybe a half hour.
Several very small feedings would be preferable to a few large ones.

Zebrafish like clean old water. Be sure
to remove any uneaten food, dead eggs and fry, and detritus from the bottom of the tank.
Do this daily if necessary to keep the dirt from accumulating on the bottom of the tank.
Zebrafish originate from fast moving rivers and streams in northeastern India and the
water quality in that area is very good.

If the water looks hazy or if you see
coleps swimming around, it is a good idea to increase the amount of water you exchange.
The haziness is probably bacteria. You should get rid of the bacteria and coleps as much
as possible. I have often changed 90% or more of the water and then if I was not
satisfied, repeated it.

It does not have to be refrigerated. Of
course, the colder temperature would reduce the growth rate of any bacteria or other
organisms that would be in it. If it is clean and nothing is growing in it, it should last
forever.

* What kind
of light do you use for your zebrafish? Do you use fluorescent light, or natural
light, or...?

Here is our
understanding of lighting and zebrafish breeding.

We keep them on a constant 14
hour on/10 hour off light cycle. The fish tend to lay their eggs within 1-2 hours of when
the lights go on.

We use fluorescent lights. We
formerly used very expensive wide-spectrum lights, but after doing
extensive tests under the illumination of different types of fluorescent lights, we found
no difference. We now use the cheapest fluorescent lights we can find.

Tests have shown that zebrafish lay
eggs best in 5-30 foot candles of illumination at the water's surface.

A frequent problem is that a room is
fitted with a timer to run the lights. There is usually a switch that can be used for
overriding the timer in case something has to be done in the dark. If someone turns this
switch on and forgets to turn it off, the fish will probably not lay eggs or will lay eggs
at unusual times. This is frequently a difficult problem to diagnose because you don't
usually go into the room when the lights are supposed to be off.

* I have a couple of beakers filled with zebrafish embryos. When will
they need some kind of aeration before they are big enough for the adult tank?

No, your baby fish in beakers do not
need to be aerated. They should not be crowded enough for oxygen depletion to become a
problem. We usually keep 20 to 30 baby fish in a beaker. I believe that the main
consideration here is not the oxygen levels, but having enough food per fish so that they
are not competing for it. A possible drawback of aerating small fish is that a too
vigorous stream of bubbles may damage the fragile babies.

Different breeders use different
methods. We are providing 2 methods below. Method #1 is the preferred method of the people
at the University of Oregon's zebrafish facility. Method #2 is the one recommended by a
high school science teacher who has successfully bred zebrafish for his science classes'
breeding program.

Method #1: One afternoon, put several egg-bearing females and a
larger number of male zebrafish into a bare tank... no gravel. The size of the tank would
depend on how many fish you are using, but a 5- or 10-gallon tank is acceptable. That
evening, put 1 or more very clean glass bowls (i.e. fingerbowls) into a corner of the
tank. These bowls should contain 2 layers of very clean marbles. Place a skirting of black
plastic around the tank and allow the fish to spend a full night in the dark. Expose the
fish to normal light the next morning and after an hour or two, remove the dishes into
which the fish will have laid their eggs. During spawning, the eggs fall among the marbles
where the adults cannot eat them. Remove the eggs from the dishes with an eyedropper or
small straw and wash them well with distilled water. Healthy fertilized eggs will appear
clear for the first day or so. Bad eggs will appear white instead of clear. This will be
apparent either immediately or after a day or so. They develop in their egg shell for a
few days before they hatch out. You may be able to see small black spots as their eyes
develop. If you have access to a dissecting microscope or a good magnifying lens you maybe
able to make out more details. They should hatch in 2-4 days. They will lay on the bottom
for a day until their swim bladders develop and then they should be swimming around and
looking for food. Raise the eggs to babies in beakers or small glass containers (100 mls
per 15 eggs). Feed the babies after they are 4 days old. At 10 days, they can be moved to
larger containers with more water.

Method #2: Use a 10 gallon tank with at least one layer of
marbles covering the bottom. The water should be at least one to two inches above the
marbles. Put a submersible heater in the tank... be sure it is completely covered with
water so it does not have hot spots exposed to the air which can damage the heater. Adjust
the temperature in the tank to between 78o and 82o F. Use NO gravel or filter in the tank.
Put an air stone or bubble wand in for aeration and water movement. One afternoon, put
several gravid (heavy with eggs) females and several more males than females in the tank.
(More males help to assure that the eggs are fertilized.) Be sure they have at least 8 to
10 hours of darkness at night since the sunrise will stimulate them to spawn. The males
will chase the females when they are mating. Mating will usually be within 1-2 hours of
when the lights turn on. Do NOT feed them now since the food will fall beneath the marbles
and decay among the eggs. Leave the adults in the tank for about 2 days. The eggs will
begin to hatch after two days and the young will begin swimming after four days. Remove
the parents by the 2nd day or they will eat the first swimmers. Once the fry are swimming
you can carefully remove the marbles and VERY carefully siphon any dirt from the bottom of
the tank. Replace any water removed from the tank with OLD aquarium water. After several
weeks when your fry are large enough, you can begin feeding them live baby brine shrimp
but be careful to wash the baby shrimp in fresh water before feeding and make sure that
the fry are large enough to eat the shrimp. Once they are feeding well, you can transfer
the babies to another larger tank with under-gravel filtration.

For beginners it is important to be aware of the value of seasons.
You should allow your fish to rest at
78oF
with 6-8 hours of light a day
for a few weeks, then increase
the
temperature to 80oF
with 8 hours illumination
per day for a week, then 82oF for 10 hours/day; finally 84oF for 12-14 hours/day,
while at the same time, increasing
the number of water changes, the quality of feed, and the quantity
of live food. All of these
conditions
help
to ensure that your
zebrafish will be in prime breeding
condition. Be sure
to keep sexes separated. When you are ready for them to breed, place
them together in tank at night. Fish will loose their prime
condition in a few weeks and they will need to be put back into the resting mode
for about a week.

For large batches I use 7
styrofoam shipping boxes available free at most tropical outlets. Green colored
nylon netting from fabric stores is draped inside the box and pinned to the top.
It helps to sew the corners together to prevent fish from getting stuck. Hang
this net a few inches above the bottom. Simply move the net (with the fish in
it) to another box every 3 days. An 18" x 18" box will hold several
hundred eggs from a dozen or more zebrafish. By the time you get back to the 1st
box (21 days), fry in that box are large enough to move easily. Feed live
blackworms (not tubifex or bloodworms) to the breeders to keep them in prime
condition. The blackworms that fall to the bottom and get through the net will
clean the water of the excess fry feed. Recommended fry feed consists of dried
peas with infusoria (available as a prepared mix commercially) which I feel
works best, followed by live brine shrimp that have been fed Selcon (obtainable
from Artemia), or at least
boiled egg yolk. The live worms do not appear to harm any fry. They do collect
all the loose shrimp eggs and filter the water well. The largest fry eat these
small worms which reproduce by splitting, instead of going after their smaller
kin. Seven boxes like this, 1 net, and 24 adult zebrafish, can easily produce a
couple hundred fry per day. To keep your breeders in top
quality condition, rest them after 21 days and replace them with fish in prime
condition. A small closet with shelves and a single space heater is all you need
to great results for very little expense.

(The above protocol was written
by Roger Hawthorne of Albany Aquarium who was the original supplier of George
Streisinger's first zebrafish models.)

* My zebrafish has gotten extremely fat. I
assume she is pregnant. I have separated her from the rest of the fish. It has been 3
weeks now, and I haven't seen any change in her. She is still "chubby." How much
longer do I wait? How long will she be pregnant for? Should I place her with the other
fish?

Zebrafish lay eggs which are fertilized
by the male after they are laid. Your fish is probably full of eggs and waiting for a male
(usually skinnier and slightly yellow on the bottom side) to mate with. She won't lay the
eggs without a male. After they lay the eggs they will tend to eat them so if you want to
keep the eggs you should either have something like marbles, rods, plants, or a mesh in
the tank that the eggs will sink into and that will keep the fish away from the eggs. The
eggs are not sticky so you can remove them with an eyedropper or maybe a straw to a clean
container in which they can hatch (in 3-5 days). It is also a good idea to get them as
clean as possible without damaging them. The fish will usually mate early in the morning
when they first see the light (sunrise or when you turn the lights on). Typically the male
fish will chase the female around. The eggs are about a millimeter in diameter (between
1/16" and 1/32") and almost clear.

* I have a breeder colony of about 60
zebrafish who have been giving me nice clutches of eggs pretty much whenever I needed
them. Suddenly yesterday and today, no eggs! Is this common?

Are you collecting eggs from all your
tanks, all the time? The fish can get exhausted if you've got them on marbles all the time
-- it's a good idea to have them on a rotating schedule. The fish can be finicky about the
light cycle. Flashing a light on them in the middle of their night can mess up their
schedules pretty thoroughly, but I wouldn't think a half-hour delay in their dawn would be
that traumatic. The most probable solution is to pull the marbles from the low fecundity
tanks to give them a rest, and to swap around males and females.

* I'm a high school senior doing a experiment concerning zebrafish breeding.
We've gotten many eggs but it is taking a lot time for us to see free-swimming fry and we
don't have a very large survival rate. Also, our eggs seem to take much longer than usual
to hatch. Could there be a reason for this?

There are at least a handful of
possible causes I can think of:

1) The temperature is too low and the
eggs are either slowed down in their development or not developing correctly due to the
cold. If your eggs are being raised in too low a temperature, they may not be developing
properly. To see this would require a dissecting scope. Fixing the problem is relatively
easy, though. Keep the eggs in a warmer place. We normally raise our eggs at 28.5O
C. (about 83O F). They can develop
at lower temperatures but they should be warmer than just normal room temperature. I would
not recommend temperatures below 24O
C. Floating a dish of eggs in your aquarium might be a way to do this for you.

2) The eggs may be contaminated and are
being killed by bacteria or fungus before they hatch. If this is the case, you should be
able to see either the bacteria or the fungus without the use of microscope. Bacteria will
make the water cloudy and smell bad. Fungus will produce a network of fine filaments that
will reach all over the dish. The solution for these problems is to better clean your
eggs. This can be done by hand with a pipette under a dissecting microscope, or with
extensive, but gentle washing in a tea strainer using clean water. A fairly good chemical
method is to lightly bleach the eggs when they are first collected. This requires a good
quality bleach (i.e. Clorox® or Purex®) rather than a cheap brand.
I have used 10 microliters of bleach in 100 mls of clean fish water for one minute
followed by three rinses in clean fish water to clean the eggs. This is a 1:10,000
dilution of the bleach, which might be easier to do than measuring 10
microliters.

3) The eggs may not be getting properly
fertilized. You could tell this by a visual inspection of the eggs. Using a dissecting
microscope, the eggs should look relatively clear (not opaque) and should be shaped like
those in a standard developmental series (such as is shown in the

Zebrafish K-12 website). If the eggs are opaque they are dead. It may
take a day for this to become apparent. This may also be apparent without a dissecting
microscope. If illuminated from below, opaque eggs will look dark since they will block
out the light. Illuminated from above, the eggs will look white since they will reflect
the light.

It is unlikely that the little
worms you describe will hurt your eggs. We sometimes notice little worms on our eggs, and
they have always proven to be one of two things: either vinegar eels (which are not eels
but actually little worms that we occasionally feed to the baby fish) or the larvae of
some harmless midges that live in the fish water. These are also a food source if the fish
find them. Neither of these worms will eat the eggs or be a health-hazard to your fish.
You won't see the vinegar eels unless you are feeding them to your fish, but the midges
seem to be able to colonize fish tanks all over the country. The actual flies they turn
into are extremely small. The larval worms are usually found on the sides or bottoms of
the tank in little tubes covered with whatever dirt-like substance is lying in the tank.

Of course, theres always the chance that yours could be some strange organism
that we havent encountered, but generally, the kinds of worms that would be a
problem for your fish would live and remain on the inside of your fish, not the outside.

Coleps are small rugby ball-shaped
ciliates (protozoans). They move in a helical (spiral) path and are covered with a series
of small plates. These creatures will sometimes contaminate your paramecia cultures.
In large numbers they can attack, kill, and completely eat you baby fish in about two
hours. (See Mazanec, A. and Trevarrow, B. (1998). Coleps, Scourge of the Baby
Zebrafish. The Zebrafish Science Monitor, University of Oregon Press, Eugene,
Oregon http://zfin.org/zf_info/monitor/vol5.1/vol5.1.html#Coleps,
Scourge of the Baby Zebrafish).

If your baby fish are disappearing "without a trace," it is likely that this
is your problem.

Thanks to the following people for providing information
for this pageDavid Badman, NIDDK, Scott Dougan, Stanford
University Medical Center, Judith H. Greenberg, NIGMS, Jerry Heindel, NIEHS, Jennifer
Matthews, University of Oregon, Bill Trevarrow, University of Oregon